Neutralizing antibodies are important effectors in humoral immune responses against bacterial or viral infections, including HIV. However, it is difficult to elicit effective neutralizing antibody responses against HIV in natural infections or by conventional vaccine approaches. Analyses the HIV viral genome and envelop protein structures have suggested that HIV has evolved many strategies to avoid neutralizing antibody responses. Currently, there are several groups of established monoclonal HIV neutralizing antibodies, which recognize the CD4 binding site, CD4-induced (CD4i) co-receptor binding site, gp120 V3 loop, or the membrane proximal external region (MPER) of gp41. Studies of these antibodies provided valuable information regarding the structural requirements for neutralizing antibodies to interact with HIV. However, a comprehensive analysis of how these neutralizing antibodies are generated is still lacking. The CD4i and the MPER antibodies have long IgH CDR3 regions with charged amino acids, which are common features of autoreactive antibodies. Indeed, two of the potent HIV neutralizing MPER antibodies 2F5 and 4E10 also react with cardiolipin and other self antigens. It has been speculated that HIV neutralizing antibodies are generated through rare recombination events and B cells expressing such antibodies have been negatively selected. Our recent results showed that the frequencies of VH replacement products are significantly elevated in IgH genes encoding anti-HIV antibodies, including anti-gp41, anti-gp120, anti-V3 loop, and CD4i antibodies. Particularly, nine of the 12 IgH genes encoding CD4i antibodies are potentially generated through VH replacement. VH replacement occurs through RAG-mediated secondary recombination involving a cryptic RSS within a rearranged VH gene and a 23 bp RSS from an upstream VH gene. VH replacement renews almost the entire VH coding region but retains a short stretch of nucleotides as a "footprint", which preferentially encodes charged amino acids to extend IgH CDR3. Almost all the VH replacement "footprints" in the CD4i antibodies contribute negatively charged amino acids into the CDR3 regions, which are critical for binding gp120. Based on these results, we hypothesize that VH replacement contributes to the B cell anti-HIV immune response. To test this hypothesis, (1) we will perform single cell PCR analysis to determine the frequencies of VH replacement products in the plasma cells of HIV patients and express recombinant antibodies to determine if these identified VH replacement products encode anti-HIV or HIV neutralizing antibodies; (2) we will use experimental VH replacement systems to generate VH replacement products with restricted usage of VH1-69 gene and determine if the artificially generated VH replacement products encode anti-HIV antibodies. Results from these studies will provide direct information regarding the contribution of VH replacement to B cell anti-HIV immune response. The current proposal is based on our recent finding that a large fraction of anti-HIV antibodies is generated through VH replacement recombination. We will use an advanced single cell PCR approach to determine the frequencies of VH replacement products in the plasma cells of HIV patients and express recombinant antibodies to determine if VH replacement products encode anti-HIV antibodies. Understanding the contribution of VH replacement to B cell anti-HIV response will have significant implications for combating HIV infection. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]